3-Acetoxymethyl-.beta.-aminoceph-3-em-4-carboxylic acid (7-ACA) is of primary importance as a starting material for the preparation of semi-synthetic commercial cephalosportns antibiotics. This intermediate is produced commercially from cephalosporin C, a fermentation product of Cephalosporin acremonium (see Newton and Abraham, Nature 175,548, 1955), by hydrolysis of the D-.alpha.-aminoadipyl side chain, either chemically or enzymatically. 3-Hydroxymethyl-7.beta.-aminoceph-3-em-4-carboxylic acid (des-7-ACA) is usually produced as an unwanted side product in the preparation of 7-ACA caused by either the chemical or enzymic (esterase) hydrolysis of the 3-acetyl group. Des-7-ACA can be produced by the action of various esterases on 7-ACA. In addition, it can be produced from desacetyl-cephalosporin C by enzymic hydrolysis in a manner analogous to the conversion of cephalosporin C to 7-ACA (World Patent No. WO 90/12110). As a result of the highly reactive nature of the C-7 amino group, chemical synthesis of 7-ACA from the deacetylated precursor results in a mixture of products. The use of typical chemical acylating reagents such as acetyl chloride or acetic anhydride produces inter alia 7-ACA, N-acetyl 7-ACA and N-acetyl desacetyl 7-ACA. In addition, 3-hydroxymethyl-7.beta.-aminoceph-3-em4-carboxylic acids are susceptible to lactonization under these conditions. Prior art methods of O-acylation involve blocking the reactive G-7 amino group, then performing O-acylation in a non-polar, organic solvent in the presence of a 4-(tertiary amino) pyridine catalyst wish an acid-acceptor base and then deblocklng the C-7 amino group (European Patent No. 153,874A). Further methods comprise of esterifying the 4-position carboxyl group of the C-7 acylated compound to prevent lactonization during the reaction, and deesterifying the carboxyl group and deprotecting the C-7 amino group to produce the 3-alkanoyloxymethyl-7.beta.-aminoceph-3-em-4-carboxylic acid (U.S. Pat. No. 3,532,694), or performing the O-acylation in an aqueous medium in the presence of a 4-(tertiary amino)pyridine catalyst with an acid-acceptor base and then deblocklng the C-7 amino group (European Patent No. 0230972). In addition, a chemical process for acetylating the 3-hydroxymethyl cephalosporins in an aqueous solvent has been described (U.S. Pat. No. 5,221,739).
Certain esterases and lipases are known to catalyze not only hydrolytic reactions but, with certain substrates, can also be used in the synthetic direction to esterify and transesterify. The use of esterases to produce desacetyl cephalosporin derivatives was first demonstrated by Jeffrey, et al. in Biochem. J., 81, 591-596 (1961) using citrus acetyl esterase (U.S. Pat. No. 3,202,656, European Patent No. 0 109 300). Subsequently, many other esterases and lipases with the ability to deacetylate cephalosporin compounds have been discovered from Aureobasidium (European Patent No. 0 044 736), Schizomycetes (U.S. Pat. No. 3,239,394), Rhizobium (U.S. Pat. No. 3,436,310), Bacillus subtilis (U.S. Pat. No. 3,304,310, European Patent No. 0 173 206), Rhodosporidium toruloides (British Pat. No. 2,060,610), Rhodotorula rubra (British Patent No. 1,474,519) and wheat germ. More recently, Wang, Y-F., et al. J. Org. Chem., 53, 3129-3130 (1988) reported the use of isopropenyl acetate as an irreversible acyl transfer reagent in the lipase-catalyzed acetylation of sugars.
We have unexpectedly discovered that use of certain enzymes, as hereinafter described, with certain acyl donors, as hereinafter described, function in the synthetic direction to produce cephalosporins acylated at the C-3' position.